Anti-idiotypic antibody and its use in diagnosis and therapy in HIV-related disease

ABSTRACT

The present invention provides an anti-idiotypic antibody having specific reactivity with an idiotope common to more than one type of anti-HIV-1 antibody, and having no specific reactivity with non-HIV-1 antibodies. The present invention provides methods of diagnosis, monitoring and treatment of HIV-related diseases through the use of this antibody or related compounds.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/610,316, filed Jul.5, 2000 (now U.S. Pat. No. 6,221,580); which is a divisional of U.S.Ser. No. 09/211,156, filed Dec. 14, 1998 (now U.S. Pat. No. 6,146,627);which is a divisional of U.S. Ser. No. 08/110,348, filed Aug. 20, 1993(now U.S. Pat. No. 5,849,583); which is a continuation-in-part of U.S.Ser. No. 07/848,327, filed Mar. 9, 1992, now abandoned, each of which isexpressly incorporated herein in its entirety by reference.

ACKNOWLEDGEMENT

This invention was made with the support of government grantsRFA-N11-1-NIAID-87-CA-03 and R01CA51434-01 from the National Instituteof Health. Therefore, the United States government may have rights inthe invention.

BACKGROUND OF THE INVENTION

This invention relates to the diagnosis and treatment of AIDS and morespecifically to an anti-idiotypic antibody reactive with more than onetype of anti-HIV-l antibody. The invention further relates to the use ofthe anti-idiotypic antibody as a diagnostic and therapeutic agent.

Acquired Immune Deficiency Syndrome, or AIDS, has been described as amodern plague. In the decade since its first description in 1981, it hasclaimed 120,000 victims in the United States alone. Currently almost200,000 people are known to be infected with the virus. However, thetrue impact of the disease has yet to be felt. The virus may remainlatent in infected individuals for five or more years before symptomsappear. Many Americans may unknowingly be infected and capable ofinfecting others who might come into contact with their body fluids.Virtually every person who contracts the virus will develop AIDS and dieas a consequence. Thus, if unchecked, the personal, social and economicimpact of AIDS will be enormous.

The causative agent of AIDS is Human Immunodeficiency Virus Type 1(HIV-1). The intact HIV-1 virion is roughly spherical and isapproximately 110 nm in diameter. The virion has an outer membranecovered with knobs or spikes made up of glycoprotein, gpl60/120. Inaddition, there exists a transmembrane protein termed gp41. Inside thevirion are two structural proteins: an outer shell composed of thephosphoprotein p¹⁷ and an inner nucleoid or central core made up of thephosphoprotein, p24. The viral RNA is present inside the core along withtwo copies of the reverse transcriptase enzyme, RT or p65, which isnecessary for the synthesis of viral DNA from the RNA template. In aninfected person, antibodies are made to each of the aforementionedprotein components and exist in characteristic concentrations throughoutthe course of the disease.

AIDS progresses through three stages after HIV-1 infection. The first isan asymptomatic stage during which the host harbors the virus, testsseropositive for HIV-1 antibodies, but does not exhibit any of thesymptoms of HIV-related disease. This stage can last for periods as longas five or more years. The second stage, AIDS-Related Complex (ARC), andthe final stage, AIDS, are symptomatic and characterized by tumors and aseries of opportunistic infections.

Shortly after HIV-1 infection a vigorous humoral response is initiated.This phase is characterized by elevated levels of circulatingantibodies. Specific neutralizing antibodies are directed against thevarious component proteins of HIV-1 and the initial virus is drasticallyreduced to levels where it is often difficult to isolate. This pointmarks the beginning of the disease-free phase of HIV infection with itshallmarks of normal T4 counts and high antibody activity against HIV-1component proteins. However, despite the presence of cellular andhumoral immunity in the infected individual, the virus persists andafter several years of latency will become active, often mutating tovariant forms, and eventually destroying the immune system leading tofull-blown AIDS.

In humans, viral infections are typically cleared by two major classesof immune response, a humoral response mediated by B-lymphocytes whichproduce antibodies and a cell-mediated immune response directed byT-lymphocytes. Individuals who test positive for HIV-1, however, areunable to clear the infection through these two types of immuneresponse. They may remain positive for several years until they succumbto the opportunistic infections characteristic of AIDS. Thus far no caseof viral clearance has been reported. The failure of the immune systemto eradicate the AIDS virus after infection remains a mystery of HIVinfection. It was first explained as a failure of the cell-mediatedresponse due to the destruction of T helper cells (T4) by HIV-1.However, this explanation is difficult to defend since the T4 countremains normal and only a small fraction of T4 cells appears to beinfected during the long latent and asymptomatic phases of the disease.

It is now considered likely that abnormalities in the humoral responseof B-cells against HIV-1 is at least in part responsible for theineffectiveness of the immune system associated with HIV-1-relateddisease. Instead of the normal polyclonal response seen in otherinfections, the antibody response in HIV-1 infected individuals appearsto result in oligoclonal or monoclonal antibody populations. Briault, etal., Clinical and Experimental Immunology 74, 182 (1988).

Traditional methods of diagnosing HIV-1 infection include serologicaltests to detect the presence of HIV-1 antibodies and polymerase chainreaction for virus detection. However, there are drawbacks to thesetraditional methods. Although they confirm the absence or presence ofHIV-1, they do not indicate the stage of disease progression. Subjectsentering the symptomatic stages of disease often fail to recognize theonset of symptoms and delay seeking help. Currently there is noeffective treatment for HIV-infection. No effective vaccine is presentlyavailable. AZT and other pharmaceutical compounds can temporarilyalleviate symptoms in AIDS patients, but have been unable to stimulatethe immune system to clear the virus.

Thus, there exists a need for further understanding of the factors whichdetermine the progress of HIV-related disease in order to provide formethods of prevention and treatment of the immune system abnormalitieswhich are characteristic of ARC and AIDS. The present inventionsatisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides compounds specifically reactive with anidiotope common to more than one type of human anti-HIV-1 antibody. Inparticular, the invention provides an anti-idiotypic antibody which isspecifically reactive with more than one type of human anti-HIVantibody, and not specifically reactive to human non-HIV antibodies. Apreferred anti-idiotypic antibody of the present invention is themonoclonal antibody designated 1F7, which is specifically reactiveagainst at least three human anti-HIV-1 antibodies of differingspecificities, and non-reactive with human non-HIV antibodies. Theidiotope recognized by the anti-idiotypic antibody of the presentinvention, and in particular the idiotope recognized by the 1F7antibody, is shown to be involved in the clonal suppression of B cellscharacteristic of HIV infection. The 1F7 antibody is also shown toinfluence T cell anti-idiotypic regulation. Therefore, the presentinvention provides a method of regulating the immune response inHIV-infected individuals through treatment with the anti-idiotypicantibody. This is thought to occur through the selective binding ofsubsets of B and T lymphocytes by the antibody of the present invention.In addition, the presence of the idiotope recognized by theanti-idiotope antibody of the present invention in the sera ofHIV-infected individuals is found to correlate with HIV-relatedpathologies, in particular HIV-related B cell lymphoma. Therefore, thepresent invention provides a method of diagnosing and monitoringHIV-related pathologies by the reaction of the HIV-infected sera withthe anti-idiotypic antibody of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the interaction of the 1F7 antibody with various HIV+ andHIV− sera or purified antibodies.

FIG. 2a shows the binding of 1F7 Id+ and F6 Id+ antibodies to gp120, and

FIG. 2b shows the binding of 1F7 Id+ antibodies to p24, while

FIG. 2c shows the binding of 1F7 to human antibodies with differentspecificities.

FIG. 3a shows the inhibition of binding of 1F7 Id+ antibodies to gp120by 1F7, and

FIG. 3b shows the inhibition of 1F7 Id+ antibodies to p24 by 1F7.

FIG. 4a shows the detection of 1F7 Id expressed on antibodies againstgp120 in HIV+ sera, and

FIG. 4b shows the detection of 1F7 Id expressed on antibodies againstp24 in HIV+ sera.

FIG. 5 shows the kappa/lambda ratio of anti-HIV antibodies, with samplenumber plotted along the y-axis and the ratio of various antibodies ineach patient plotted along the x-axis.

FIG. 6 shows the kappa/lambda ratio of affinity purified anti-p24antibodies captured by p24 ELISA or 1F7 ELISA for patient 3-P14 (6a) andpatient 3-P49 (6b).

FIG. 7 shows the IEF pattern of purified anti-p24 and anti-gp120antibodies.

FIG. 8 shows histograms of DNA content on HIV-(8A and 8C) and HIV+ (8Band 8D) donor PBMC cells cultured with 1F7 or a control antibody.

FIG. 9 shows 1F7 induced apoptosis on PBML cell cultures from HIV+donors compared with apoptosis induced with a control antibody.

FIG. 10 shows the comparison of spontaneous apoptosis with 1F7 inducedapoptosis on PBML cell cultures from HIV+ donors.

DETAILED DESCRIPTION OF THE INVENTION ABBREVIATIONS

HIV, human immunodeficiency virus

HIV-1, HIV type 1 virus

HIVIG, human polyclonal anti-HIV immunoglobin

IVIG, pooled human polyclonal immunoglobin

Id, idiotope

Ab, antibody

Id+ Ab, idiotype positive antibodies

V, variable regions of antibody

VH, variable heavy chain

Vk, variable kappa light chain

Vl, variable lambda light chain

gp120, HIV-1 envelope glycoprotein

p24, HIV core protein (gag)

RT, p65, HIV-associated reverse transcriptase

gp160, HIV-1 whole envelope glycoprotein

gp41, transmembrane HIV-associated protein

As used herein, the term “HIV-related disease” refers to both theasymptomatic and symptomatic phases, that is the ARC and AIDS phase,which follow HIV-1 infection. The terms “AIDS” and “ARC” refer toAcquired Immune Deficiency Syndrome and AIDS-Related complex,respectively, as described by Adler, British Medicine, 294:1145 (1987),which is herein incorporated by reference. AIDS is characterized bytumors and a series of opportunistic infections. As used herein, theterm “antibody” refers to any molecule which has specificimmunoreactivity activity, whether or not it is coupled with anothercompound such as a targeting agent, carrier, label, toxin, or drug.Although an antibody usually comprises two light and two heavy chainsaggregated in a “Y” configuration with or without covalent linkagebetween them, the term is also meant to include any reactive fragment orfragments of the usual composition, such as Fab molecules, Fab proteinsor single chain polypeptides having binding affinity for an antigen. Fabrefers to antigen binding fragments. As used herein, the term “Fabmolecules” refers to regions of antibody molecules which include thevariable portions of the heavy chain and/or light chain and whichexhibit binding activity. “Fab protein” includes aggregates of one heavyand one light chain (commonly known as Fab), as well as etramers whichcorrespond to the two branch segments of the antibody Y (commonly knownas F(ab)₂), whether any of the above are covalently or non-covalentlyaggregated so long as the aggregation is capable of selectively reactingwith a particular antigen or antigen family.

As used herein, the term “idiotope” or idiotypic determinant refers toan antigenic determinant or epitope unique to the immunoglobulin productof a single clone of cells. The idiotope is found in the variable regionof the antibody. The term “epitope” refers more generally to anantigenic determinant on a molecule which is recognized by antibodies.The term “anti-idiotypic” or “anti-idiotypic antibody” refers to anantibody raised against a first antibody which specifically binds to anidiotope of the first antibody.

As used herein, the term, “specificity” is used interchangeably with theterms “specific reactivity” “specifically reactive” and“immunoreactivity”. Each term refers to a binding affinity which isgreater than background binding. For example, a binding affinity that ismeasured by an optical density of greater than the standard deviation ofthe mean optical density of a control as determined by conventionalELISA techniques is considered to represent specific reactivity. Otherassays known in the art may also be used to determine specificreactivity. The term “not significantly reactive” is a binding affinitywhich is not greater than background binding.

As used herein the term “clonotype” refers to the homologous product ofa clone of cells, or the phenotype of a clone of cells. Idiotypicdeterminants expressed by populations of antigen-specific B cells or Tcells serve as clonotypic markers for immune cells responding to a givenantigenic challenge. Clonotypic specificities may be linked to a singleepitope specificity or may be shared by antibodies of differentspecificities (Zhou, et al. J. Immun. 145, 2554 (1990)).

The present invention provides a compound which is specifically reactivewith more than one type of human anti-HIV-1 antibody, and notspecifically reactive with human non-HIV-1 antibodies. In particular,the present invention provides an anti-idiotypic antibody which isspecifically reactive against an idiotope common to more than one typeof human anti-HIV antibody, and not specifically reactive with humannon-HIV antibodies. A preferred anti-idiotope antibody is the monoclonalantibody 1F7, which specifically reactive with a common clonotype sharedby anti-HIV-1 antibodies having different specificities.

The anti-idiotypic antibody of the present invention is useful as adiagnostic and prognostic marker to measure the level of reactive HIV-1antibodies in samples from HIV-infected patients. Analysis of theselevels allows early characterization of HIV-related disease progress,allowing the early diagnosis and treatment of HIV-related pathologies.In addition, the anti-idiotypic antibody is useful therapeutically tofacilitate viral clearance. Dominant B-cell clones producing antibodieswhich express the idiotope against which the antibody is reactive andwhich are no longer effective against HIV-1 are suppressed, therebyrestoring the normal polyclonal immune response which can effectivelyclear the virus from the infected individual's system.

The anti-idiotypic antibody of the present invention is produced againstHIV-infected sera, and then screened for specific reactivity againstantibodies reactive against various HIV antigens, and for lack ofreactivity against human non-RIV antibodies. The anti-idiotypic antibodyis specifically reactive with antibodies reactive against different HIVantigens, such as anti-p24 antibodies, anti-gp120 antibodies and anti-RTantibodies (Id+ antibodies).

The antibody of the present invention may be produced by methods wellknown in the art. For example, Fabs and methods for making them aredescribed in Harlow and Lane, Antibodies, A Laboratory Manual, 626-631(1988), which is incorporated herein by reference. Examples of reactivesingle chain polypeptides and a method to generate them are taught byLadner, U.S. Pat. No. 4,946,778, which is incorporated herein byreference. Antibodies may be produced by monoclonal techniques such asthe method of Kohler and Milstein, Nature 256, 495 (1975), which may bemodified by Gerhard, Monoclonal Antibodies, Kennett et al., eds.,370-371 (1980), for example, both of which are incorporated herein byreference. Alternatively, antibodies can be screened from polyclonalselections by well-known assay methods. Antibodies may also be producedby recombinant DNA techniques as taught by Cabilly et al., U.S. Pat. No.4,816,567, for example, which is herein incorporated by reference, orselected from immunoglobulin combinatorial libraries as taught by Huse,et al., Science 246, 1275 (1989), which is also incorporated byreference. CDR grafting as taught by Cabilly et al., supra, can also beused to produce fragments reactive with the idiotope.

In addition, the invention provides an isolated idiotope found to becommon to several types of HIV-1 antibodies (ID+ antibodies),particularly those produced by dominant restricted B-cell clones, andspecifically reactive with the anti-idiotypic antibody of the presentinvention. This idiotope, when recognized and isolated from an ID+antibody, or otherwise synthetically or recombinantly produced, isuseful in the production of monoclonal and polyclonal antibodies fordiagnostic, prognostic and therapeutic applications using techniqueswell known in the art. For example, the isolated idiotope may be used toimmunize animals to generate hybridomas expressing monoclonal antibodiesspecific for the idiotope. In other cases, the idiotope may be used tostimulate an immune response in a rabbit, goat, non-human primate orother animal from whose serum polyclonal antibodies may be obtained bymethods well known in the art. In addition, the idiotope may be used forthe purification or characterization of anti-idiotypic antibodies ofinterest, for example, monoclonal antibodies or antibodies present inhuman tissue or body fluids.

In addition to antibodies, the methods of the present invention canemploy non-antibody compounds which have a binding affinity for theidiotope recognized by the antibody of the present invention, and whichdo not bind to non-HIV antibodies. Such compounds can be constructedaccording to methods known in the art and include peptide or non-peptidedrugs, for example, which have a specific binding affinity for therecognized idiotope.

The present invention provides a method of restoring oligoclonality inthe antibody response to HIV infection through the administration of theanti-idiotypic antibody or other reactive compound of the presentinvention to an HIV+ individual. Anti-idiotypic regulation can enforceoligoclonality either by suppressing the dominant B cell response to HIVor reinforcing the growth of other clones. This approach to treatment isbased on the particular characteristics of HIV infection.

During the time HIV-1 inhabits the host, its major neutralizingepitopes, particularly gp120, undergo rapid mutations giving rise toneutralization-escape variants. Nara, et al., Journal of Virology 64,3779 (1990); Nara, et al., FASEB Journal 5, 2437 (1991). It is thoughtthat the antibodies which were produced in response to the initial HIV-1infection are not effective against the new escape variants and that thehost immune system fails to produce new antibodies to the variants.Montefiori et al., Virology 182, 635 (1991); Nara et al.

J of Virology 64, 3779 (1990); Nara et al., PNAS USA 84, 797 (1987). Thefailure of the immune system to recognize these newly-emerging virusisolates is thought to be due to the establishment of a dominant clonalpopulation of B-cells committed, or restricted, to producing antibodiesto the original HIV-1 variant. The dominant clones prevent therecruitment and expansion of other B-cell populations which could becapable of an effective response to new HIV-1 variants and otheropportunistic viruses. In other settings, dominantly established B-cellshave been found to exert a suppressive effect on minor B-cells which aredirected against the same or cross-reactive epitopes. Askonas andWilliamson, Nature 238, 339 (1972); Briles and Davie J Exp Med 152, 151(1980).

It appears that as HIV-1 generates neutralization escape variants, themutated epitopes retain sufficient cross-reactivity to continue totrigger the early, clonally expanded B-cell clones. Evidence for thiscross-reactivity with envelope epitopes has been observed in divergentisolates. Kang et al., PNAS USA 88, 6171 (1991). Thus, B-cells withpotentially higher affinity for mutated epitopes may be limited in theirability to respond and proliferate to reach an effective clone size.Instead, cross-stimulation by mutated HIV-1 epitopes continues totrigger and expand the original and now ineffective clones. Recentreports on constant IEF profiles over time in infected individualsdocument the persistence of anti-gp120 specific B-cell clones.Matricardi, et al., Seventh International Conference for AIDS 2, 167(1991). By this mechanism a situation can develop in which the infectedindividual produces human neutralizing antibodies that are effectiveagainst laboratory strains and isolates from different seropositiveindividuals, but less effective against the autologous HIV-1 variants.Montefiori et al. sunra, (1991); Nara, et al., supra (1990).

The approach to treatment of HIV infection provided by the presentinvention is based on the surprising discovery that idiotope common tomore than one type of HIV-1 antibody is produced by restricted B-cellclones. Clonal restriction of B-cells producing these antibodies hasbeen demonstrated by kappa/lambda light chain analysis, isoelectricfocusing, and immunoblot analysis of variable region diversity of B-cellcolonies responding to HIV-1 infection, as demonstrated in Example IIIbelow.

A preferred anti-idiotypic antibody is the monoclonal 1F7 antibody. Theproduction and screening of 1F7 is described in Example 1 below. Ahybridoma cell line capable of expressing the 1F7 antibody has beendeposited on Mar. 8, 1993 with the American Type Culture Collection,10801 University Boulevard, Manassas, Va., 20110-2209, and has beenassigned ATCC No. HB 11286.

1F7 is found to be specifically reactive with at least three anti-HIVantibodies having separate specificities. It is shown in Example IIbelow that the 1F7 Id is a clonotype marker for anti-HIV antibodies. Aclonotypic analysis of HIV+ and HIV− sera demonstrates that it is commonfor the 1F7 Id clonotype to be shared by different anti-HIV-1 antibodiesproduced by the same HIV+ individual (see Western blot, FIG. 1, andTABLE 2). Because a shared clonotypic marker on B cells responding toHIV infection would be expected to be the target of regulation andmaintenance of the immune response to the HIV virus, the 1F7 Id isconsidered to be involved in anti-idiotypic regulation of the immunesystem in HIV-infected persons.

Evidence is provided that anti-HIV+ antibodies and 1F7 ID+ antibodies inparticular are of restricted clonal origin. This is demonstrated by thedata shown in Example III A, B, and C below.

Therefore, the present invention provides a method of overcoming thedominance of the ineffective clones by blocking the idiotope recognizedby the anti-idiotypic antibody of the present invention. Treatment of apatient with the compound of the invention will result in the specificbinding of the antibody or related compound to the idiotope on cells inthe dominant clones. This results in the inhibition of theirproliferation and curtailment of the production of antibodies to theoriginal HIV-1 variant. As a result of the dominant cells being blocked,other B-cells are able to respond to the new HIV-1 variants. A normalpolyclonal immune response is restored and antibodies to the new HIV-1variants are effective in clearing the virus from the host system.

It is further shown that T cells in HIV+ infected persons may be subjectto anti-idiotypic regulation via the idiotope recognized by 1F7. T cellsfrom HIV+ sera sample were analyzed for evidence of anti-idiotypicregulation by the 1F7 anti-idiotypic antibody as described in Example Vbelow. Peripheral Mononuclear Blood Cells (PMBC) were taken from serafrom HIV+ individuals and HIV− normal control individuals and grown inculture according to the methods described in Example V below. Thesecells were exposed to the 1F7 antibody and an isotype control antibody.It was found that the 1F7 antibody induced apoptosis in 16 of the 20HIV+ samples, and none of the HIV− control samples. Flow cytometricanalysis and lymphocyte subset depletion experiments described inExample V below demonstrated that cells undergoing apoptosis were the Tcells subset CD8+ cells. These findings indicate that 1F7 binds a subsetof T-lymphocytes selectively expanded in HIV infection through receptormolecules linked to cell-mediated apoptosis. In addition, theexperiments described in Example VI below demonstrate that 1F7 reducesT-cell mediated cytotoxicity in HIV+ sera. Because CD8+ cytotoxicT-lymphocytes (CTL) are known to circulate in HIV-1 infected individuals(Walker et al., Nature 328, 345 (1987)), a reduction in CTL activity inresponse to 1F7 is another indication that 1F7 is capable of influencingthe composition of T cells in HIV-infected individuals. Therefore, byselectively binding to subsets of B and T lymphocytes expanded duringHIV infection, the antibody of the present invention is expected tofavorably alter the immune response in HIV infection.

In another aspect of the present invention, the presence of the idiotoperecognized by the antibody of the present invention in a sample of humansera is found to correlate with the occurrence of HIV-relatedpathologies in the individual. As described in Example IV, therecognition of the 1F7 idiotope on IgG and IgM correlates with the HIV+condition, and in particular correlates with the occurrence of HIV-1related lymphoma. Studies show that 93% of HIV-1 infected patients withlymphoma test positive for 1F7 expression. Therefore, the detection ofthe 1F7 idiotope by reaction with the 1F7 antibody is a method ofpredicting, detecting, or monitoring the occurrence of HIV-1 relatedlymphoma in HIV+ patients. The detection and diagnosis of the lymphomaat an early stage is particularly useful because it allows for a moreaggressive and suitable treatment of this particular malignancy.

The invention further relates to use of the antibody or non-antibodycompound of the present invention to detect certain types of HIV-1antibodies in a sample. This can be useful for identifying HIV-1antibodies in cells or other samples being prepared for laboratory use,or in samples from patients suspected of having HIV-related disease. Oneembodiment involves contacting the sample suspected of containing HIV-1antibodies with the antibody or compound of the present invention anddetermining whether binding occurs. For example, a sample from a patientmight be serum, semen, urine, tissue, cells or any other samplegenerally known or suspected to contain antibodies. A sample might alsobe spent culture from cell lines being tested for HIV-1 antibodyproduction. The antibody is most easily put in contact with the sampleby mixing them together in a test tube or in the depression well of aslide. Subsequent determination of binding may be performed by any meansknown in the art, for example, radioimmune assay, staining of labelledcompound, or immunoprecipitation. Binding affinity above backgroundindicates the presence of HIV-1 antibodies and is diagnostic ofHIV-related disease.

In addition, the invention relates to the use of the antibody ornon-antibody compounds of the present invention to obtain a quantitativedetermination of HIV-1 antibody level in order to prognose a patient'sHIV-related disease progression so that effective therapy can beinitiated. As used herein, the term “prognose” of “prognosing” refers tomonitoring the progression, determining the prognosis or forecasting theappearance of symptoms of HIV-related disease. Previous methods ofplotting T4 cells and antibody levels have proven unsatisfactory as soleprognostic tools. The levels of HIV-1 antibody obtained by practicingthe present invention are the result of the reliable, progressivedevelopment of clonal dominance of B-cell colonies. The antibodiesproduced by these colonies exhibit the novel idiotope and bind to theantibody or compound of the invention. Quantitative results regardingbinding may be obtained as described above. The amount of bindingdetermined by the procedure is compared to a standard for the variousstages of HIV-related disease. In this way, it is possible to determinethe stage of the patient's disease and prognose the development offurther symptoms. The standard could be prepared, for example, by themethods described above using fluid samples from patients with symptomsknown to characterize specific stages of HIV-related disease anddetermining the amount of HIV-1 antibody specifically reactive with theantibody or Id-reactive compound of the invention. Resulting levels ofantibody are charted against time correlating to stage of progression.The resultant chart serves as the standard on which results frompatients of unknown diagnosis and prognosis are plotted. The stage ofprogression and prognosis of the patient's disease is determined bycomparison of his level of HIV-1 antibodies which react to the antibodyor non-antibody compound compared to the standard. The 1F7 antibody ofthe present invention, for example, has been shown, for example, to beparticularly precise in monitoring HIV-related lymphoma.

The invention further relates generally to methods for the prevention,immunization and treatment of HIV-related disease by administering aneffective amount of the compound to a patient diagnosed with HIV-relateddisease. An effective amount of antibody to treat HIV-related disease isthe amount necessary to produce the effect of destroying clonaldominance of restricted B-cells or restricted or infected T cells, thusrestoring a polyclonal immune response in the patient. The compound maybe provided as a pharmaceutical composition containing an effectiveamount of the compound together with a physiologically acceptablecarrier. This composition may be administered by any means known in theart, for example, intravenous, intramuscular, subcutaneous, intradermal,intraperitoneal, or by infusion. The compound may also be bound to asuitable therapeutic agent such as a toxin, hormone, drug or othercompound to facilitate the destruction of dominant B-cells. Drugsinclude, in general, alkylating agents, antiproliferative agents,tubulin-binding agents, cytotoxins, and the like. Toxins suitable astherapeutic agents include podophyophyllotoxins, ricin, thetrichothecenes, the colchicenes and pseudomonas endotoxin.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed.

EXAMPLE I Isolation and Characterization of Anti-idiotypic Antibody 1.Materials

Human polyclonal anti-HIV immunoglobulin (HIVIG), lot VH 102 wasobtained from NIAID AIDS Research Reference Reagent Program (ERCBioservices Corporation, Rockville, Md.). Human pooled IgG (IVIG) waspurchased from Cutter Biological, Elkhart, Idn. Normal human HIV-serawas obtained from the San Diego Regional Blood Bank. HIV positive (HIV+)sera from healthy, seropositive individuals were obtained from NorthAmerican Biological Inc. (NABI), Miami, Fla.

Recombinant p24 (HTLV-IIIB) and the p24/gp41 fusion protein wereobtained from Dr. Torsten Helting, Pharmacia Genetic Engineering, LaJolla, Calif. Recombinant gp120 (HTLV IIIB), or gp120 (MN) V3 looppeptides, and recombinant HIV-1 reverse transcriptase (RT, p65) werepurchased from American Biotechnologiest Inc., Cambridge, Mass.Recombinant gp120 (SF2) was obtained from Chiron Corporation,Emeryville, Calif. The antigen designations IIIB, MN, SF2, etc. refersto laboratory strains of HIV-1.

Human monoclonal anti-HIV-1 antibodies were previously described in thepublications of Gorny et al., PNAS USA 88, 3228 (1991), and Gorny etal., PNAS USA 86, 1624 (1989), which are both herein incorporated byreference. These include human monoclonal anti-p24 antibodies, 71-31,91-5, and 241-D (all IgG1, lambda), anti-gp120 antibodies, 257-D, 268-D,and 453-D (all IgG1, lambda) and anti-gp41 antibody 98-6 (IgG2, kappa).

2. Methods 1. Production of Mouse Monoclonal Antibody Against HumanPolyclonal HIVIG

Monoclonal antibodies were produced according to the method of Muller etal., J Immun 147, 933 (1991), which is herein incorporated by reference.BALB/c mice (MTS Laboratories, San Diego, Calif.) received threesubcutaneous injections of 50 μg of human HIVIG in Freund's complete andincomplete adjuvant (Sigma Chemical Co., St. Louis, Mo.) on days 1, 10and 20. On day 45 the mice were boosted with 50 μg HIVIG intravenously.Mouse splenic cells were then fused with Sp2/0 cells according tostandard protocols. Culture supernatants were screened as describedbelow.

Microtiter plates were coated with 100 μl/well of 2 μg/ml of eithergp120 or p24 in bicarbonate buffer (pH 9.6) at 4° C. overnight, andblocked with 2% BSA for two hours following by adding 100 μl/well of 100μg/ml of HIVIG and incubated for two hours. After washing three timeswith 0.01M PBS containing 0.1% Tween-20 (PBS-T), 100 μl of hybridomaculture supernatants were added and incubated for two hours. Afterwashing another three times, 100 μl of 10⁵ cpm ¹²⁵I-labeled goatanti-mouse Ig were added to each well and incubated at 37° C. for 1.5hours. The plates were washed three times and cut for counting todetermine cpm bound to each well in the gamma counter (Nuclear-MedicalLaboratories, Inc.).

Alternatively, replicas of the microtiter plates were coated with 100μl/well of 2 μg/ml of IVIG (Cutter Biological, Elkhart, Ind.) at 4° C.overnight, then blocked with 2% BSA. IVIG, which refers to human pooledIgG from human sera not containing HIV antibodies, which is generallyderived from a large donor pool of greater than 10,000 donors. Thesubsequent procedure of adding culture supernatants and ¹²⁵I-labeledgoat anti-mouse Ig was the same as described above. Hybridoma antibodieswhich bound to antibodies captured by HIV antigens, not to IVIG, wereselected.

Positive clones were subcloned four times and expanded in tissue cultureflasks or amplified by ascites in BALB/c mice. Monoclonal antibodies ofIgM isotype derived from supernatants and ascites were purified on goatanti-mouse IgM Sepharose 4B column (Pharmacia LKB, Biotechnology AB,Uppsala, Sweden).

3C1, another IgM kappa clone from the same fusion as produced 1F7, isused as a negative control in several experiments.

2. Western Blot Analysis

Western blot analysis was performed using commercial HIV-1 ImmunoblotKits according to manufacturer's instruction (Bio-Rad, Hercules,Calif.). For detecting human anti-HIV antibodies, human sera or1F7-purified human antibodies were incubated with nitrocellulose stripscontaining HIV-1 antigens for 30 minutes. After washing, the antibodiesthat bound to HIV antigens were detected using alkalinephosphatase-conjugated goat anti-human IgG. For detection of 1F7positive anti-HIV antibodies captured by HIV antigens, thenitrocellulose strips were first incubated with human sera for 30minutes. After washing two times, 3 ml of 1F7 supernatants were added toeach strip, and incubated for 2 hours. After repeatedly washing, eachstrip was incubated for one hour with 3 ml of peroxidase conjugated-goatanti-mouse IgM (1:200 dilution, Fisher Biotech, Pittsburgh, Pa.). Thedevelopment of the strips was performed using 3 ml of 600 μg/ml ofdiaminobenzidine (DAB) (Sigma Chemical Co., St. Louis).

3. Affinity Purification of Human Serum Antibodies

Immunoaffinity purification of human serum antibodies was performed bycoupling recombinant p24 protein, recombinant p24/gp41 fusion proteinand mouse anti-idiotypic antibody, 1F7, respectively to CNBr-activatedSepharose 4B (Pharmacia LKB, Biotechnology AB, Uppsala, Sweden)according to the manufacturer. The purification of anti-phosphorycholine(PC) and F6 Id+ antibodies (F6 idiotope positive antibodies) was carriedout as described in Halpern, et al. J. Clin. invest. 88, 476 (1991),which is herein incorporated by reference. F6 is an unrelatedanti-idiotypic antibody which reacts with natural human anti-PCantibodies, and is used as a control in experiments involving 1F7. Inorder to obtain anti-gp41 antibodies on a p24/gp41 affinity column, HIV+serum was first absorbed repeatedly on p24 affinity column to removeanti-p24 antibodies totally. The flow-through was then passed overp24/gp41 immunoabsorbent.

Sera from HIV+ individuals was diluted with 0.01M PBS, pH 7.0, andapplied to immunoabsorbents. After extensive washing with PBS, boundantibodies were eluted with 0.1M glycine buffer (pH 2.5), dialyzedagainst PBS and concentrated.

4. ELISA for Detection of Human Antibodies to qp120 and p24

The recombinant gp120, RT, or p24 were coated in microtiter plates at100 μl/well of 2 μg/ml overnight at 4° C., then blocked with 2% BSA.Human sera or purified human antibodies diluted in 0.01M PBS containing0.1% Tween 20 (PBS-T) and incubated for 2 hours. Wells were thenincubated with peroxidase conjugated-goat anti-human IgG (1:5000, TAGO,Inc., Burlingame, Calif.) for one hour. All incubations were followed bywashing five times with PBS-T. Bound antibodies were visualized usingO-phenylenediamine (OPD)(Sigma Chemical, St. Louis), and the reactionwas stopped with 3N H₂SO₄. Optical density (OD) was read at anabsorbance of 490 nm (Molecular Devices Corporation, Menlo Park,Calif.).

5. ELISA for Determination of 1F7 Id Expression on Anti-p24 , Anti-gp120Antibodies, and Anti-RT Antibodies

This procedure was similar to the HIV antigen-capture RIA for detectionof anti-HIV antibodies described above. Microtiter plates were coatedwith 200 ng/well of gp120 (IIIB or SF2), RT, or p24 at 4° C. overnight,then blocked with 2% BSA. Human sera (1:100 dilution) oraffinity-purified human antibodies were added to each well and incubatedfor 2 hours. After washing, 100 ng/well 1F7 supernatants or 3C1 (acontrol mouse monoclonal IgM) supernatants were added and incubated for2 hours, followed by additional washing. 1F7 or 3C1 bound to antibodiescaptured by gp¹²⁰, RT or p24 were detected with peroxidaseconjugated-goat anti-mouse IgM.

6. ELISA for Competitive Inhibition of Binding of 1F7 ID+ Antibodies togp120 and p24 by 1F7.

The procedure of coating, washing, and developing of microtiter plateswas performed as described above, except that the 1F7 ID+ antibodies,which were purified by 1F7 iumunoabsorbent from HIV+ serum werepre-incubated with inhibitors at 4° C. overnight before being added tothe plates coated with gp120 or p24. The percent of inhibition wascalculated as follows:${\% \quad {inhibition}} = {100 \times \left( {1 - \frac{{OD}\quad {with}\quad {inhibitor}}{{OD}\quad {without}\quad {inhibitor}}} \right)}$

7. ELISA for Identification of Human Anti-HIV Antibodies Binding with1F7

Microtiter plates were coated with 500 ng/well of 1F7 at 4° C. overnightand blocked with 2% BSA for two hours. 100 μl of diluted human sera orhuman antibodies purified by affinity chromatography, were added to eachwell and incubated for two hours at room temperature. After washing fivetimes, the plates were incubated with peroxidase conjugated-goatanti-human IgG for 1.5 hours.

Alternatively, 100 μl/well of 1 μg/ml of Biotin-labeled 1F7 were addedand incubated for one hour. The plates were washed another five timesand then 100 μl/well of avidin-labeled peroxidase (1:2000, SigmaChemical, St. Louis) was added and incubated for 45 minutes. Subsequentprocedure of this assay is as described above. Biotinylation of 1F7 wasperformed as described by Harlow and Lane, eds. Antibodies, A LaboratoryManual (1988), which is herein incorporated by reference.

Procedure

Mouse monoclonal anti-idiotypic antibodies were prepared as describedabove against HIVIG. Antibodies against HIVIG were detected using p24and gp120 capture ELISA assays. One hybridoma, 1F7 (IgM, kappa) whichbound to antibodies captured by these HIV-1 antigens, but not to IVIGwas subcloned and grown as ascites fluid as described above.

Testing using HIVIG and IVIG antibody capture ELISA using p24 and p120antigens as shown in TABLE 1 showed that sera from HIVIG immunized micebound to HIVIG captured by p24 or gp120 and also to IVIG, whilepre-immune sera did not bind to HIVIG or IVIG. Supernatant from 1F7,however, only bound to HIVIG captured by HIV-1 antigens. The binding of3C1 was used as a negative control.

TABLE 1 p24-HIVIG gp12-HIVIG IVIG pre-immunized sera 300 400 450post-immunized sera 24000 21600 22300 1F7 10400 6300 550 3C1 670 690 600

Western blot analysis was then performed. Three different reactivityprofiles were distinguished using sera from different HIV+ individuals,as shown in FIG. 1. #14 HIV+ serum produced several bands in the p18,p24, gp41 and p55, p65 and gp16 positions when reacted with 1F7supernatant. #14 HIV+ serum antibodies were purified on 1F7immunoabsorbent as described above and also analyzed by Western blot.Antibodies purified by 1F7 immunoabsorbent showed binding patterns verysimilar with that produced by this serum with 1F7. Serum and 1F7purified from #3 HIV+ serum showed bands only in the p24 and p55regions. #16 HIV+ serum showed no bands. All three HIV+ sera producedbands with multiple EIV-1 antigens when nitrocellulose strips weredeveloped with alkaline phosphatase-conjugated goat anti-human IgG. FIG.1 is as follows: lane 1: HIV+ patient #14 serum, developed with goatanti-human IgG; lane 2: HIV+ patient #14 serum, incubated with 1F7 anddeveloped with goat anti-mouse IgM; lane 3; Spg of 1F7 affinity purifiedantibody from patient #14, developed with goat anti-human IgG. lane 4:HIV+ patient #3 serum, developed with goat anti-human IgG; lane 5: HIV+patient #3 serum, incubated with 1F7 and developed with goat anti-mouseIgM; lane 6: 5 μg of 1F7 affinity purified antibody from patient #3,developed by goat anti-human IgG. lane 7: HIV+ patient #16 serum,developed with goat anti-human IgG; lane 8: HIV+ patient #16 serum,incubated.with 1F7 and developed with goat anti-mouse IgM; lane 9: HIVnegative control serum, developed with goat anti-human IgG; lane 10: HIVpositive control serum, developed with goat anti-human IgG. All humansera were diluted 1:100.

Since the Western blot analysis shown in FIG. 1 showed binding of 1F7 toanti-core and anti-envelope antibodies from #14 HIV+ serum, theseantibodies were purified from #14 HIV+ serum by 1F7 immunoabsorbent asdescribed above. The absorbed material on the column was eluted withacid to yield 1F7 Id+material. Eluted material was concentrated andtested for binding to p24 and gp120 in an ELISA as described above. InFIG. 2a the binding of 1F7 ID+ antibodies to insolubilized gp120 isshown. For a control, #14 HIV+ serum was absorbed on an unrelatedanti-ID, F6. Binding to insolubilized gp120 is shown in FIG. 2b. In FIG.2c, eluted Ig from the p24 affinity column binds strongly toinsolubilized 1F7. The flow through from the p24 column was absorbed onand eluted from a chimeric antigen p24/gp41 affinity column. Onlyresidual binding of anti-gp4 antibodies to 1F7-coated plates wasobtained indicating that the majority of 1F7 ID+ antibodies are p24specific in this serum. As a control, anti-PC antibodies purified fromHIV+ serum to 1F7 were employed, as seen in FIG. 2c. To obtain theresults shown in FIG. 2, microtiter plates were coated with 200 ng/wellof gp120 or p24 and then incubated with human antibodies purified by 1F7or F6 immunoabsorbent. FIG. 2c shows the binding of 1F7 to humanantibodies with different specificities. 1F7-coated plates wereincubated with human antibodies purified by HIV antigen or PC affinitycolumn. The antibodies bound to 1F7 were detected with biotinylated 1F7as described above. All human antibodies were purified from #14 HIV+serum.

FIGS. 3a and 3 b show inhibition of binding of purified 1F7 ID+antibodies to gp120 and p²⁴ respectively by 1F7. Plates were coated witheither gp120 or p24. 1F7 ID+ antibodies were added together withdilutions of 1F7 or a control mouse IgM monoclonal antibody, 3C1, asdescribed above.

EXAMPLE II Clonotypic Analysis of HIV+ and HIV− Human Serum

According to the Western blot show in FIG. 1 described above, three 1F7reactivity patterns with HIV+ sera were observed. In one group,represented by #14 HIV+ serum, 1F7 reacted to antibodies to more thanone HIV antigen. In the second group, represented by #3 HIV+ serum, 1F7showed reactivity only with anti-core (p24) antibodies. Serum from thethird group showed no 1F7-positive anti-HIV antibodies.

Further testing was performed in order to analyze the representation ofthese three groups in HIV+ sera. The detection of anti-gp120 andanti-p24 antibodies, and in addition, the detection of 1F7 Id+ onantibodies against gp120 and p24 in HIV+ sera was determined as follows.First, anti-HIV antibodies were detected as described above, by coatingmicrotiter plates with 200 ng/well of gp120 or p24. A 1:40 dilution ofHIV+ individual serum was added and incubated. The antibodies whichbound to gp120 and p24 were determined by peroxidase conjugated-goatanti-human IgG. For detection of binding of 1F7 to anti-HIV antibodies,the plates coated with gp120 and p24 were first incubated with 1:40diluted HIV+ sera, then 1F7 supernatants were added and incubated. Thebinding of 1F7 to anti-HIV antibodies captured by gp120 or p24 wasdetected with peroxidase conjugated-goat anti-mouse IgM. Boundantibodies were visualized using O-phenylenediamine (Sigma, St. Louis,Mo.), as described above, and the optical density read at 490 nm.

The results are presented in FIG. 4. FIG. 4a shows the anti-gp120antibodies and anti-gp120/1F7 antibodies detected for each of the 15HIV+ samples. FIG. 4b shows the anti-p24 and anti-p24/1F⁷ antibodiesdetected for the same 15 HIV+ sera samples. In the 15 HIV+ sera samples,5 show anti-p²⁴ and anti-gp120 antibodies. In two sera, only anti-p24antibodies are 1F7 reactive, and in another 2 sera anti-gp120 antibodiesare 1F7 positive. The remaining six HIV+ sera are negative for 1F7.These data indicate that it is not uncommon for the 1F7 Id clonotype tobe shared by different antigen-specific antibodies.

To further corroborate the prevalence of idiotope sharing anti-HIV-1antibodies, HIV-1 antigen capture ELISA, as described in Example 1, wasused to detect expression of 1F7 Id on different anti-HIV-1 antigens insera from 40 HIV-1+individuals. This procedure is described in Wang etal., Eur. J. Immunol. 22 1749 (1992), which is herein incorporated byreference.

Microtiter plates were coated with p24, gp120 (IIIB or SF2), RT andtetanus toxoid (used as a control, obtained from Pharmacia), all 200ng/well respectively, then blocked with 2% BSA. HIV-1+ sera diluted1:100 with PBS were added and incubated for 2 hours. After washing, theplates were incubated with 1:5000 diluted peroxidase-conjugated goatanti-human IgG for 1.5 hour. Bound antibodies were visualized using OPD(o-phenylenediamine) at 490 nm. The ELISA for 1F7 Id was similar. Afterincubation of HIV-1+ sera (1:100) with plates coated with HIV-1 antigensand tetanus toxoid, 100 ng/well of 1F7 were added, and incubated foranother 2 hours. After washing, the plates were incubated with 1:2000peroxidase-conjugated goat anti-mouse IgM for 1.5 hour. The subsequentprocedure was the same as above.

As shown in Table 2, among 40 HIV-1+ sera, 9 (22.5%) show anti-p24,anti-gp120 and anti-RT antibodies, all being 1F7+. In 3 (7.5%) sera, twoof three kinds of anti-HIV-1 antibodies are 1F7 Id+, and in 11 (27.5%)sera, only one kind of antibody is 1F7+. Altogether, 57.5% of HIV-1+sera have at least one kind of anti-HIV-1 antibody which expresses 1F7Id. These data confirm that it is common for the 1F7 Id clonotype to beshared by different anti-HIV-1 antibodies produced by the sameindividual.

TABLE 2 No. positive Percent Antibody sera 1F7Id 1F7 Id⁺ Total anti-p2430 18 60 Total anti-gp120 36 16 44.4 Total anti-RT 15 12 80 Totalanti-tetanus toxoid 39 0 0 Anti-p24 + anti-gp120 + anti-RT 0 22.5Anti-p24 + anti-gp120 1 2.5 Anti-gp120 + anti-RT 1 2.5 Anti-p24 +anti-RT 1 2.5 Anti-p24 or anti-gp120 or anti-RT 11 27.5 Total 23 57.5

EXAMPLE III Evidence that Anti-HIV Antibody Responses in HIV+Individuals are of Restricted Clonal Origin A. Determining ClonalRestriction of B-Cell Clones by Kappa/Lambda Light Chain Analysis 1.Methods

Purification of human serum antibodies was performed by couplingrecombinant p24 (HIV-1 IIIB) (Pharmacia Genetic Engineering, La Jolla,Calif.) and recombinant gp120 (SF-2) (Chiron Corporation, Emeryville,Calif.) to CNBr-activated Sepharose 4B (Pharmacia LAB, Biotechnology AB,Uppsala, Sweden). According to the manufacturer, IgG from HIV+ sera(North American Biological, Inc., Miami, Fla.) was purified on protein Gsepharose. The Ig fraction was passed over affinity columns at 5ml/hour. After washing, the column was eluted using 0.1M glycine buffer(pH 2.5) with 0.01 M PBS (pH 7.0). The antibodies were dialyzed andconcentrated for further use.

Detection of antibody light chain isotypes in normal (San Diego RegionalBlood Bank, San Diego, Calif.) and unfractionated HIV+ human sera (NorthAmerican Biological, Inc., Miami, Fla.) was performed by coatingmicrotiter plates with goat anti-human IgG (TAGO, Inc., Burlingame,Calif.) at 100 μl/well of 1 μg/ml in 0.05M bicarbonate buffer (pH 9.6)overnight at 4° C. and blocked with 2% BSA for three hours at roomtemperature. Sera was diluted 1:20,000 in 0.01M PBS containing 0.1%Tween 20 (PBS-T) and incubated for two hours at room temperature. Afterwashing three times with PBS-T, 100 μl of peroxidase conjugated goatanti-human kappa or lambda antibody (TAGO, Inc., Burlingame, Calif.)diluted 1:4,000 and 1:3,500, respectively, were added and incubated for1.5 hours at room temperature. After washing, bound antibodies werevisualized using O-phenylenediamine (OPD) (Sigma Chemical, St. Louis)and reaction was stopped with 3N H₂SO₄. Optical density (OD) was read atan absorbance of 490 nm (Molecular Devices Corporation, Menlo Park,Calif.). The kappa/lambda ratio was calculated according to theequation: ratio=OD of kappa/OD of lambda.

Detection of antibody light chain isotypes of purified anti-HIVantibodies was performed by coating microtiter plates with 5 μg, 2 μg or1 μg of each recombinant gp120 (SF-2), gp120 (IIIB), p24, or RT (IIB) in0.05M bicarbonate buffer (pH 9.6) overnight at 4° C., then blocked with2% BSA for three hours at room temperature. Human sera diluted 1:100 inPBS-T were added and incubated for two hours at room temperature.Subsequent procedures were the same as described above.

Detection of light chain isotypes of anti-id1F7 were performed bycoating microtiter plates with 100 μl/well of 5 μg/ml of 1F7 inbicarbonate buffer overnight at 4° C., then blocked with 2% BSA forthree hours at room temperature. 100 μl diluted human sera or humanantibodies purified by HIV-1 antigen affinity chromatography were addedand incubated for two hours at room temperature. Subsequent steps arethe same as described above.

2. Results

In human sera, polyclonal immunoglobin usually consists of near equalamounts of kappa and lambda light chains. Over-representation of eitherlight chain isotype is characteristic of monoclonal or oligoclonalantibody populations, as described in Mdller et al., J Immun 147, 933(1991). Forty randomly selected sera from HIV-1 infected individuals andforty normal sera were assayed by ELISA for binding to light chainisotype specific antisera as described above. When the kappa/lambdaratio was calculated none of the unfractionated HIV+ sera showedevidence of a monoclonal gammopathy.

The same kappa/lambda ratio analysis was then performed with antibodiesderived from sera of forty different seropositive individuals specificfor four different HIV-1 antigens. Antibodies to p24(IIIB), gp120 (2SF),gp120 (IIIB) and RT(IIIB) were captured by the correspondinginsolubilized antigens. In contrast to the equivalent light chainisotype representation in total Ig, the showed either preferential kappaor lambda usage as represented in FIG. 5. FIG. 5 shows the kappa/lambdaratio for each patient was calculated as described above and plotted onthe y-axis. 90% of the HIV+ sera had a skewed kappa/lambda ratio,demonstrating that oligoclonal or monoclonal anti-HIV antibodies arecommon in the sera of HIV+ individuals.

Subsequently, plates were coated with the 1F7 antibody and sera fromHIV+ individuals added and reacted with either anti-kappa or anti-lambdaantisera. Kappa and lambda light isotypes were both detected on 1F7captured antibodies. To test whether 1F7 is associated with restrictedanti-HIV antibodies, anti-p24 antibodies from different sera werepurified by affinity chromatography as described in Example I. Bothpurified antibodies were then allowed to bind to p24 and 1F7 coatedplates and the ratio of kappa to lambda calculated. As shown in FIG. 6a,ten times more kappa reactivity than lambda was found when assayed onp24 plates. The kappa/lambda ratios was significantly less skewed whenassayed on 1F7 coated plates. This was also demonstrated for FIG. 6b,using sera from a second HIV+ individual. Collectively, this datademonstrated that anti-p24 responses consist of two populations ofantibodies: one being clonally restricted and the other polyclonal.

B. Determining Clonal Restriction of B-cell Clones by IsoelectricFocusing 1. Method

Isoelectric focusing was performed using precast pH 3-10 gel accordingto the manufacturer (Novex, Novel Experimental Technology, San Diego,Calif.). The Novex system utilized a cathode buffer composed of 0.29%(w/v) arginine and 0.35% (w/v) lysine whereas the anode buffer consistsof 0.47% (w/v) of phosphoric acid (85%). Anti-p24 and gp120 (SF-2)antibodies purified by affinity chromatography were adjusted to a finalprotein concentration of approximately 100 μg/ml with sample bufferprovided by Novex, 20 μl were applied to the gel. The Novex model 3540power supply was programmed to produce three phases of constant voltage(i.e. 100 v for one hour followed by 200 v for one hour and, finally,500 v for thirty minutes). The run time was for a total of 2.5 hours.The current per gel was set at a maximum of 8 mA. Visualization ofisoelectric focusing separated proteins was by silver staining (Bio-RadLaboratories, Richmond, Calif.).

2. Procedure

To further demonstrate restricted character of anti-HIV antibodyresponses, the anti-p24 anti-gp120 antibodies with biased light chainisotypes were purified on affinity chromatography and submitted toisoelectric focusing. As seen in FIG. 7, anti-p24 and anti-gp120antibodies show IEF bands typical for oligoclonal or monoclonalantibodies. The anti-p24 antibodies contains two IEF restricted bandingclusters one at pH 8.5 to 9.6, and the other at pH 7.2 to 7.4. Theanti-gp120 antibody shows one cluster at pH 7.0 to 7.5.

C. Determining Clonal Restriction of B-cell Clones by ImmunoblotAnalysis of Variable Region Diversity 1. Method of identifying V_(K) andV_(H)

Immunodominant portions of the first framework region of Ig heavy andlight chains were used for the creation of anti-peptide antisera thatare specific for the V_(k) and V_(H) gene families according to themethods of Silverman et al., (Arthritis and Rheumatology 33,1347 (1990),Journal of Immunological Methods 95, 249 (1986), Journal of ClinicalInvestigations 88, 911 (1991)), all of which are herein incorporated byreference. Antisera were also used that identify sequences from thefirst domains of heavy and light chain constant regions. Briefly,peptides were conjugated by their carboxyl-terminal cysteine to thecarrier, keyhole limpet hemocyanin, prior to immunization of rabbits. Todemonstrate the binding specificity of the peptide-induced reagents,Western immunoblot analyses were performed with 4 μg/lane of Igproteins.

2. Procedure

Antibodies were affinity purified on p24 and gp120 immunoabsorbentcolumns respectively and used to analyze their variable regionexpression. The kappa/lambda analysis was performed on the sera used forpurification, taken from four HIV+ patients, identified as 3-P14, 3-P49,P14, and P15. Ig chains were electrophoretically separated prior totransfer to membranes that were then probed with anti-peptide antibodiesspecific for Ig constant or variable region sequences. For each patientthe total unfractionated IgG is compared to the affinity purifiedantibodies to HIV-1 gp120 and p24, and these compared to the monoclonalIgM proteins KAS (V_(H1)-V_(K3)) and HER (V_(H3)-V_(K3)) which serve ascontrols for subgroup specificity. The replicate panels were tested withantiserum specific for first framework sequences specific for the fourV_(K) families, and the major V_(H) families, V_(H1), V_(H4) and V_(H3).These families represent the overwhelming majority of circulating Ig andB cells in normal adults according to Berman et al., Embo. J 7, 727(1988), Logtenberg et al. Int. Immunol. 1, 362 (1989), Guigou et al.Mol. Immunol. 27, 935 (1990), and Zouali et al. J. Immunol 146, 2855(1991). The antiserum to Ig constant regions (C_(H), C_(K) and Cλ) areused as controls. Immunoblots reveal that unfractionated IgG from normalcontrols and the four HIV-1 infected individuals contain subpopulationsfrom all of these V region families. In contrast, the anti-p24antibodies are all depleted of V_(H3) derived H chains and enriched forVH1 derived H chains. Only one individual (3-P49) also had enrichment ofanti-p24 antibodies from the VH4 family. Anti-gp120 antibodies were alsostudied in three of these individuals (3-P14, 3-P49, P14). One of theseindividuals (p14) also had enrichment in V_(H1) derived H chains anddepleted in V_(H4) and V_(H3) H chains, while a second individual(3-P14) was found to have anti-gp120 antibodies with enrichment ofV_(H1) and V_(H4) H chains, with depletion in V_(H3) derived H chains.Patient 3-p49 also has V_(H3) depleted in anti-gp120 antibodies.Analysis of the L chains revealed that both lambda and kappa L chainsare used, but no consistent pattern of V_(k) family usage was detected.The biased light chain isotype expression of anti-p24 antibodies fromfour selected sera was confirmed in Western blots using V_(k) familyspecific antisera. Three out of four antibodies showed enrichment forV_(KII) and depletion in lambda activity. The data on V gene familyutilization and isotype expression are compiled in TABLES 3 and 4. Thekappa/lambda and 1F7 ratios are determined by ELISA as described inExample 1 above. The 1F7 binding is determined by goat antimouseIgM-HRPO, and substrate and O.D. measured at 490 nm. Subgroup analysiswee performed with replicate immunoblots of isolated H and L chainsusing variable region family specific, anti-peptide antibodies asdescribed above. Relative loading of H chains and L chains weredemonstrated with antisera to gamma and Ck constant regions determinantsrespectively.

The results shown in TABLES 3 and 4 indicate clonal restriction inanti-HIV antibodies and are in agreement with the results obtained byIEF on purified anti-p24 and anti-gp120 antibodies.

TABLE 3 Immunoblot VH reactivity of affinity purified anti-HIVantibodies SUBGROUP ANALYSIS Gamma VH1 VH3 VH4 κ/λ 1F7+ Anti-p24 3-p14+++ ++++ − − 14.29 1.593 3-p49 ++ ++ − +++ 0.09 0.235 p14 ++ ++ − − 5.961.750 p15 +++ ++++ + + 2.86 0.786 Anti-gp120 3-p14 ++++ +++ − ++++ 0.250.568 3-p49 ++++ ++++ +++ ++++ 0.62 — p14 ++++ ++++ + +++ 0.61 1.043 p15N.D. N.D. N.D. N.D. 0.87 — Anti-p24 antibodies* anti-p24 (+) ++ +++ + +anti-p24 (−) +++ ++++ ++++ ++++ N.D. = Not Done +,++,+++,++++, indicatesrelative band intensity −, indicates not detected *refers to affinitypurified anti-p24 antibodies (eluate) compared to non-anti-p24antibodies (flow through) from sera of HIV+ patients.

TABLE 4 Immunoblot VL reactivity of affinity purified anti-HIVantibodies SUBGROUP ANALYSIS Ck VkI VkII VKIII VkIV Cl Anti-p24 3-p14++++ ++++ ++++ +++ + − 3-p49 ++ − − ++ + +++ p14 + + +++ + ++ − p15 +++++ +++ +++ ++ + Anti-gp120 3-p14 ++++ + − +++ ++ +++ 3-p49 ++++ +++ +++++++ ++++ ++++ p14 ++++ ++ ++++ +++ + ++++ Anti-p24 antibodies* anti-p24(+) ++ + ++++ ++ ++++ ND anti-p24 (−) ++++ ++++ ++++ ++++ ++++ ND *, seeTable 3.

Collectively, the data on light isotype expression, IEF and V geneutilization provide strong evidence that the anti-HIV antibody responsesin seropositive individuals are of restricted clonal origin.

EXAMPLE IV Correlation of Presence of IF7 Idiotope with VariousHIV-related Disorders

A comparison of IF7 idiotope expression in sera from various HIVinfected and noninfected patients was conducted. The amount ofcirculating IF7 positive immunoglobulin was measured in the followinggroups: seropositive B cell lymphoma patients, asymptomatic seropositiveindividuals, symptomatic seropositive patients (ARC and AIDS), andseronegative individuals including seronegative lymphoma patients.

A. Material and Methods 1. Sera

Sera from B cell lymphoma patients without HIV-1 infection were providedby San Diego Regional Cancer Center. Sera from HIV-1 virus infectedpatients, AIDS, ARC, and HIV-1 related lymphoma were provided from theAIDS study group of San Francisco General Hospital. Sera from 28 HIV-1infected asymptomatic individuals and ARC/AIDS patients were provided bythe Southwest Foundation for Biomedical Research, San Antonio, Tex. HIVseronegative and seropositive sera were purchased at the local bloodbank.

2. Production of Mouse Monoclonal Anti-Idiotypic Antibody 1F7

Splenic cells from BALB/c mice immunized with human polyclonalanti-HIV-1 IgG were fused with Sp2/0 cells according to standardprotocols as described -in Example I above. Antibodies binding to HIVIGwere detected using p24 and gp120 (IIIB) capture ELISA. One hybridoma(1F7, IgM), which bound to antibodies captured by HIV-1 antigens, not toIVIG and IVIG captured by non-HIV antigens, was subcloned and grown asascites in BALB/c mice. Ascites fluid was purified on goat anti-mouseIgM Sepharose 4B column (Pharmacia LKB, Biotechnology AB, Uppsala,Sweden).

3. ELISA to Detect 1F7 on Human IgG and IgM

Microtiter plates were coated with 100 ng/well goat anti-mouse IgM at 4°C. overnight and blocked with 2% BSA. 100 ng 1F7 was added to each welland incubated at room temperature for 2 hours. After washing, 1:1000diluted HIV-1+ or HIV-1− human sera was added to each well and incubatedfor another 2 hours. Subsequently, either peroxidase conjugated goatanti-human IgG (1:6000, Fisher Biotech, Pittsburgh, Pa.) or peroxidaseconjugated goat anti-human IgM (1:1000) were added for 1.5 hours. BoundIF7+ antibodies were visualized using o-phenylenediamine OPD (SigmaChemical, St. Louis, Mo.) and the reaction was stopped with 3 N H₂SO₄.Absorbance was read spectrophotometrically at 490 nm (Molecular DevicesCorporation, Menlo Park, Calif.).

4. Statistical Methods

Statistical analysis to calculate the significance of differencesbetween groups was undertaken using a t-test for unpaired variables. Forcalculation of predictive values, sensitivity and specificity, theNewman-Keuls Multiple Comparison Test was used.

B. Results

The expression of the 1F7 idiotope in sera from HIV infected andnoninfected patients is shown below in TABLE 5. Data is indicated as themean of the log of the optical density at 490 nm×1000, together with thecalculated standard deviation in each patient group. It can be seen thatthe highest correlation occurs for HIV+ related lymphoma.

It was also established that the elevation of 1F7-Id in polyclonalantibodies in HIV+ lymphoma patients was not due to a variation of totalIg. Total immunoglobin in sera from randomly selected patients withARC/AIDS or AIDS related non-Hodgkin B cell lymphoma (AIDS-NHL) wasdetermined by standard procedures in the clinical reference laboratoryof the San Francisco General Hospital. Total Ig in 10 sera from theARC/AIDS cohort (that is, group of statistically equivalent individuals)and in 10 sera from the AIDS lymphoma patient cohort was found to be inthe same range. In addition, it was established that the 1F7 marker wasmore than a marker for the longevity of the disease, since the levels of1F7-Id expression in sera were not found to be correlated with thedecrease of the CD4 cell count. CD4 cell count considered to be aparameter for disease progression from asymptomatic stages of HIV-1infection to AIDS. A survey of 1F7-Id expression in sera from 10 HIV+individuals with a CD4 cell count higher than 400 cells/mm³, sera from10 patients with a CD4 cell count between 200 and 400 cells/mm³, andsera from 8 patients with a CD4 cell count lower than 200 cells/mm³revealed no significant differences in titers of antibodies expressing1F7 idiotope.

TABLE 5 EXPRESSION OF 1F7 IDIOTOPE ON IgG AND IgM IN HIV-1+ AND HIV-1−SERA # of Group sera IgG IgM HIV + Lymph 44 2.60 +/− 0.50 1.77 +/− 0.61ARC 76 1.73 +/− 0.59 1.15 +/− 0.48 AIDS 75 1.56 +/− 0.64 1.25 +/− 0.41Asymptomatic 90 1.41 +/− 0.65 1.32 +/− 0.52 HIV− 60 1.10 +/− 0.41 0.89+/− 0.32 HIV − Lymph 20 0.82 +/− 0.38 0.71 +/− 0.04

It can be seen that the highest correlation for the expression of 1F7 isobtained for HIV+ lymphoma.

Therefore, a statistical analysis of 1F7+ idiotope expression in serafrom patients with HIV-1 related lymphoma was performed. 1F7 idiotopeexpression in HIV-1 related lymphoma was compared to all other HIV+groups (pooled). This can be seen in TABLE 6 below. 1F7 idiotopeexpression on IgG antibodies is highly specific (95%) and highlysensitive (90%) in patients with HIV related lymphoma. The positivepredictive value for lymphoma in HIV-1 infected individuals wascalculated to be 0.755.

TABLE 6 PREDICTIVE VALUE, SENSITIVITY AND SPECIFICITY OF 1F7 IDEXPRESSION IN HIV-1 RELATED LYMPHOMA Statistical 1F7 + Id on 1F7 + Id onParameter IgG Ab IgM Ab Pos. Predictive Value 0.755 0.295 Neg.Predictive Value 0.984 0.935 Sensitivity 90.9% 70.5% Specificity 95.0%71.6%

In the survey described above and shown in TABLE 6 there is strongevidence that the described HIV-1 associated idiotope 1F7 serves as amarker for HIV-1 related B-cell lymphoma. 93% of HIV-1 infected patientswith lymphoma tested positive for 1F7 expression. Significantly lowerlevels of 1F7 idiotope were detected in HIV-1 infected patients withoutlymphoma.

According to the calculated statistical parameters as shown in TABLE 6,high levels of 1F7 Id 40 expression can predict AIDS associated lymphomain 75% of cases. This strongly suggests that the 1F7 idiotope can beused as a disease marker.

EXAMPLE V Enhancement of Apoptosis In HIV+ Patients BY 1F7 A. Materialsand Methods 1. 1F7 Stimulation of PBML Cell Culture

Blood from 20 HIV sero-positive donors was obtained from the Universityof California San Francisco hospital from Michael McGrath. Blood from 8HIV sero-negative donors were collected from laboratory workers. Wholeblood from donors was collected, and clotting prevented by treatmentwith citrate according to methods known in the art. Plasma was collectedafter centrifuging at low speed and stored frozen until anti-1F7 levelswere tested. Peripheral blood mononuclear lymphocytes (PBML) wereisolated from whole blood by centrifugation on “Lymphoprep” (Nycomed,Norway), according to manufacture's suggestions. PBMLs were collectedfrom the top of the Hypaque cushion, and washed free of human serum byrepeated spins in “Growth Medium” (RPMI-1640 plus 10% (V/V) fetal calfserum (Hyclone, USA) including 1 mM L-Glutamine, 1 mM Sodium-Pyrovate,1/100 non-essential amino acids (all GIBCO), 50 mM HEPES (SIGMA), pH7.4). The cells were plated out at a concentration of 2.5×10⁶/ml in 2ml/well of a 24 well tissue culture plate (Costar). Cells werecultivated in Growth Medium in the presence of 5 μg/ml mouse IgMantibody, either MOPC-104E (Sigma, St. Louis, MO) or TEPC 183 (isotypecontrol antibody, Sigma, St. Louis, Mo.) or 1F7 (isolated from hybridomasupernatant or ascites fluid, as described above). Cells were harvestedafter cultivation for 1, 3, 7 or 10 days for determination of levels ofapoptosis.

2. CD4⁺ and CD8⁺ Depletion

CD4⁺ or CD8⁺ cell free fractions were generated by negative selectionfrom the PBMLs of HIV sero-positive donors. These donors werearbitrarily selected among donors with CD4⁺ cell counts above 500.Magnetic beads, commercially available as Dynabeads (DYNAL), labeledwith either anti-CD4 antibodies or anti-CD8, were incubated with PBMLsat a ratio of 3:1, beads to target cells, at 4° C. for 30 minutes undergentle rotation end over end. Beads, and cells bound to beads wereisolated by means of attraction to a strong magnet according to themanufacturer's direction. The remaining cells were collected. Extractionsuccess was determined by flow cytometry analysis of the remainingpopulation. Extraction was always better than 95%. This population wassubsequently cultivated and subjected to apoptosis determination.

3. Double Staining with FITC and PI

Cells were harvested from culture and stained with fluorescein(FITC)-conjugated antibodies to CD4 and CD8 (Gentrak, Plymouth Meeting,Pa.). FITC-conjugated murine isotype (Gentrak) was used as a stainingcontrol. After staining, the cells were resuspended in propidium iodide(20 μg/ml in 0.112% sodium citrate) (Sigma, St. Louis, Mo.) andincubated for 30 minutes at room temperature prior to cytometricanalysis.

Flow cytometric analysis was carried out on a FACScan (Becton Dickinson,San Jose, Calif.). Two-parameter cytograms of red fluorescence (DNAcontent) rsus green fluorescence (FITC) were generated to determine thepercentage of cells that were CD4 or CD8 positive.

4. Quantitation of DNA Degradation (Apoptosis) by Propidium Iodide (PI)

Cells were harvested from culture and fixed with 70% ETOH for a minimumof 3 hours. After 2 washes cells were resuspended in PI (50 μg/ml PI,180 U/ml RNase in PBS)(Sigma, St. Louis, Mo.) and incubated for 30minutes at room temperature prior to flow cytometric analysis.

Flow cytometry was performed on an Epics I (Coulter Electronics,Hialeah, Fla.) using doublet discrimination in the measurement of DNAfollowed by a MULTICYCLE (Phoenix Flow Systems, San Diego, Calif.)software program. The percentage of DNA to the left of the G₀/G₁ peakwas taken to represent DNA lost from the original 2N quantity as aresult of apoptotic degradation.

B. Results 1. 1F7-Induced Apoptosis of PBMLs from HIV+Donors

A typical example of PBMC (peripheral blood mononuclear cells) from anHIV seropositive donor undergoing 1F7-induced apoptosis is shown in FIG.8 (panel B and D) after an in vitro incubation of PBMC for 7 days. FIG.8 is a histogram of DNA content on HIV-(histogram A and C) andHIV+(histogram B and D) donor PBMC after 7 day culture with either mouseIgM (MOPC-104E) or 1F7. Data was generated after fixation and stainingwith PI as described above. Histograms A and C show no apoptosis in thePBMC of HIV− healthy control donor as measured by the amount of degradedDNA. The onset of 1F7-induced apoptosis in PBMC derived from thispatient could be observed after 24 hours (FIG. 9). The level ofspontaneous apoptosis was compared to 1F7 induced apoptosis by PBMCsfrom 20 different HIV+ donors after 7 days of culture (see FIG. 10). 1F7significantly increased the level of apoptosis for 16 donors (>3%increase), whereas cells from 4 donors were not affected. The averagenumber of spontaneous to 1F7 induced cells in apoptosis is 19% and 32%respectively. Apoptosis of PBMLs from 8 HIV− donors were unaffected by1F7. There was no significant correlation between spontaneous apoptosislevels and degree to which 1F7 induced apoptosis.

2. 1F7 Induced Adoptosis in PBMLs Depleted of CD4+or CD8+Cells

In order to determine if the observed apoptosis induced with 1F7 wasassociated with cells of a particular phenotype, PBMLs from a HIV+ donorwere separated into a CD4⁻ population and a CD8⁻ population by negativeselection. The effect of 1F7 on these populations were compared to apreparation of unseparated PBMLs from the same donor (see Table 7).PBMLs from an HIV sero-positive donor were divided into three pools: Oneremained complete, the second was depleted of CD4⁺ cells and the thirdwas depleted of CD8⁺ cells. Subsequently each pool was cultivated for 3or 7 days with either 5 μg/ml of a control antibody, MOPC-104E (Sigma,St. Louis, Mo.), or 5 μg/ml 1F7 at what point the level of apoptosis wasdetermined by flow cytometry. Table 7 below shows that 1F7 inducedapproximately 4× control levels of apoptosis in the CD4⁻ populationearly after start of culture. This in contrast to 1F7 induced apoptosisin the CD8⁻ population was less severe, and slower, approximately 2times less than control levels.

TABLE 7 Percent Apoptosis Culture Conditions Complete CD4⁻/CD8⁺CD8⁻/CD4⁻ Day 3 Control 13 11 17 1F7 16 41 17 Day 7 Control 4 4 17 1F7 55 29

In summary, it was found that the 1F7 antibody induced apoptosis in 16of the 20 HIV+ samples, and none of the HIV− control samples. Apoptoticcells appear in the A0 regions with a DNA below that of the G0/G1 cells.Flow cytometric analysis and lymphocyte subset depletion experimentsdemonstrated that cells undergoing apoptosis were T cell subset CD8+cells.

EXAMPLE VI 1F7 Influence on T Cell-Mediated Cytotoxicity

In addition to the enhancement of apoptosis in HIV+ patients by 1F7,experiments indicate that 1F7 reduces T-cell mediated cytotoxicity.Because CD8+ cytotoxic T-lymphocytes (CTL) are known to circulate inHIV-1 infected individuals, a reduction in CTL activity in response to1F7 indicates that 1F7 is capable of influencing the composition of Tcells by reducing the number of CD8+ cells. The following experimentswere performed according to the methods described in Grant et al., AIDS6, 1085 (1992), which is herein incorporated by reference.

In one experiment, the addition of 1F7 at the start of cell culturereduced T cell-mediated cytotoxicity in IL-2 stimulated PBMC cultures,as measured by anti-CD3 mediated lysis of P815 cells. The experiment wasperformed as follows. Peripheral blood lymphocytes from 3 HIV+individuals (same source as Example V) were cultured at 1×10⁶/ml inmedia plus lpg/ml of either 1E7 or an IgM isotype control MOPC-104E(Sigma,). After 3 days, recombinant IL-2 (Genzyme, Cambridge, Mass.) wasadded at 5 units/ml. After 4 days in IL-2, the lymphocytes were testedfor total CTL activity by redirected lysis of anti-CD3 coated P815 cellsaccording to Grant et al., supra. Anti-CD8 coated P815 cells were usedas a negative control and were not killed. It was found that cellscultured with 1F7 experienced a reduction in percent CTL-mediated lysiswhen compared with the control of between approximately 35 percent toapproximately 20 percent to approximately 50 percent of the control,depending on the effector to target ratio, which was varied from 25:1 to12.5:1 to 6.25:1. The CTL-mediated lysis assays were performed accordingto Grant et al., supra, at page 1087.

In a second set of experiments, incubation of CTL-derived from HIV+individuals with 1F7 immediately before standard ⁵¹Cr release assays(Grant et al., supra, at page 1087) also reduced lysis of specifictargets. The specific targets in this case were EBV (Epstein-Barrvirus)-transformed B cells infected with various recombinantvaccinia/HIV constructs, as described in detail in Grant et al., supra,at page 1087. Anti-HIV CTL were generated by stimulating peripheralblood lymphocytes from an HIV-1 infected individual with autologousPRA-activated lymphocytes. After 3 days of stimulation, recombinant IL-2(Genzyme, Cambridge, Mass.) was added at 5 units/ml for an additional 7days before cells were tested. Autologous EBV-transformed B cellsinfected at a MOI of 15 overnight with vaccinia recombinants describedabove expressed either individual HIV proteins or b-galactosidase as acontrol. Immediately before asays 1×10⁶ effector cells were incubatedfor 45 minutes with 1 μg/ml of either 1F7 or MOPC-104E (Sigma, St.Louis, Mo.). After incubation, effector cells were washed twice andtested for cytolysis of autologous EBV-transformed B cells infected atan MOI of 15 overnight with vaccinia recombinants expressing HIV gag,pol or env or E. coli β-galactosidase (lacZ) as a control. Cellsincubated with 1F7 had a reduced percent specific lysis of approximately30 percent of the control lysis depending on the effector: target ratio.

Although the invention has been described with reference to thepresently-preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

We claim:
 1. A method of restoring oligoclonality in the immune responseto HIV-1 infection comprising administering a sufficient amount of ananti-idiotypic monoclonal antibody or binding fragment thereof havingspecific reactivity with an an idiotype specifically bound by monoclonalantibody 1F7 produced by hybridoma ATCC Accession No. HB 11286, andwhich idiotope is common to at least three types of humananti-HIV-1-antibody of differing specificities, but not significantlyreactive with idiotypes of human non-HIV-1 antibodies.
 2. The method ofclaim 1 wherein the monoclonal antibody which is designated 1F7 isproduced by hybridoma ATCC Accession No. HB
 11286. 3. The method ofclaim 1, wherein the idiotope is a clonotype of human polyclonalanti-HIV-1 antibodies.